Investigating Interferon Antagonists in Delaying Innate Immune Responses to SARS-CoV-2

TITLE: Investigating Interferon Antagonists in Delaying Innate Immune Responses to SARS-CoV-2
PI: Susan C. Baker, PhD, Loyola University Chicago Stritch School of Medicine
The goal of this proposal is to determine how viral interferon antagonists function in the replication and
pathogenesis of coronaviruses, particularly during replication of Severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2). Coronaviruses (CoVs) are a diverse family of positive-sense RNA viruses that
include pathogenic strains infecting human and animal hosts. CoVs have repeatedly jumped from animal
reservoirs into human circulation, causing severe disease and pandemics, as we are currently experiencing
with SARS-CoV-2. Developing appropriate protective measures against emerging CoVs, including SARS-CoV-
2, will depend upon gaining an understanding of coronavirus-host interactions. We discovered that the
endoribonuclease (EndoU), a highly conserved component of the CoV replicase complex, reduces dsRNA
species recognized by host pattern recognition receptor MDA5, delaying the induction of interferon. We
reported that viruses expressing an inactive form of EndoU replicate as efficiently as wild type virus in IFN non-
responsive cells. Importantly, replication of EndoU mutant CoVs in interferon-responsive cells activate robust
immune responses, which extinguishes virus replication and reduces pathogenesis in animals. Recently, we
identified the target of EndoU activity to be poly-uridine containing negative sense RNA, which we term PUN
RNA. This removal of the PUN RNA delays the generation of dsRNA species that are recognized by host
pattern recognition receptor MDA5. We hypothesize that EndoU activity contributes to the delay in the
innate immune response to SARS-CoV-2 replication. Here, we propose to investigate the mechanism of
how EndoU acts in SARS-CoV-2, how EndoU associates with the replicase complex, and how PUN RNA
contributes to activating MDA5. In Aim 1, we will evaluate EndoU and other IFN antagonists for their role as
modulators of Type I and Type III IFN responses to SARS-CoV-2 infection in primary human airway cells and
in enterocytes. We will use reverse genetics to generate viruses with inactive IFN antagonists and evaluate the
effects of combining inactivation of EndoU with inactivating mutations of other viral protein IFN antagonists. In
Aim 2 we will delineate and disrupt EndoU interactions within the coronavirus replicase complex. The results of
these studies will guide strategies for disruption of EndoU from the CoV replicase complex, which would
activate protective immune responses to CoV infections. In Aim 3, we will identify regions of poly-uridine
negative-sense RNA, termed PUN RNA, required for recognition by EndoU and MDA5. These studies will
provide new information on how PUN RNAs are recognized by EndoU and MDA5. Overall, these studies will
define a new mechanism for how an endoribonuclease acts as a virulence factor. This new information can be
used to develop antiviral therapies and vaccines against existing and emerging coronaviruses.

Grant Number: 5R01AI159945-05
NIH Institute/Center: NIH
Principal Investigator: Susan Baker